The natural regulation of thrombin by its principal native serine proteinase inhibitors (serpins), antithrombin and heparin cofactor II, is thought to become overwhelmed in thrombotic disorders. Although these plasma proteins, in particular in the presence of heparan sulphate or dermatan sulphate glycosaminoglycan cofactors, inhibit thrombin in a suicide substrate mechanism that is likely physiologically irreversible, their affinity for thrombin is much lower than that of the leech protein, hirudin. The hirudins are a family of closely-related small proteins, 65 to 66 amino acids in length, that bind thrombin with sub-picomolar inhibitory constants. As such, they constitute the most potent known polypeptide inhibitors of thrombin.
The potency of hirudin as a thrombin inhibitor originally raised high hopes that it would become a mainstay of interventional cardiology, and other medical areas with unmet clinical needs for antithrombotic and adjunctive anticoagulant agents. Currently, however, recombinant hirudins produced in yeast, such as desirudin and lepirudin, are primarily used in heparin-induced thrombocytopenia, and not in acute coronary syndromes, where extensive investigation failed to show superiority over heparin in primary outcomes. Hemorrhagic complications narrow the therapeutic window of hirudin and limit its applications. Its pharmacokinetics are also a challenge to optimal application. Hirudin's small size and tightly disulphide-bonded, compact structure result in rapid, renally mediated clearance from the circulation. In the laboratory of the present inventor, the clearance of hirudin in animal models has been altered by fusion to albumin (Syed S, Schuyler P D, Kulczycky M, et al., Blood 1997; 89: 3243-3252), and it was demonstrated that lower doses of the fusion protein had anticoagulant and antithrombotic effects greater, or at least more durable, than its smaller predecessor. Nevertheless, the fusion protein still promoted bleeding, and lowering the dose in combination with an antiplatelet protein-albumin fusion only modestly reduced this tendency (Sheffield W P, Gataiance S, Eltringham-Smith L J., Thromb Res 2007; 119: 195-207).
Other efforts to regulate hirudin's potent antithrombin activity have centred on converting it into a prodrug. Extrapolating from the known importance of a free N-terminus for maximal effectiveness of hirudin (Lazar J B, Winant R C, Johnson P H., J Biol Chem 1991; 266: 685-688), a recombinant single chain anti-fibrin antibody fused at its light chain C-terminus to the N-terminus of hirudin, via a factor Xa cleavage site was produced (Peter K, Graeber J, Kipriyanov S, et al., Circulation 2000; 101: 1158-1164). However, the fusion protein had little or no antithrombin activity unless exposed to factor Xa. Similarly, tri- or tetrapeptide factor Xa, factor XIa, or thrombin cleavage sites were added to the N-terminus of hirudin, temporarily blocking its activity, until procoagulant protease cleavage restored its function (Zhang C, Yu A, Yuan B, et al., Thromb Haemost 2008; 99: 324-330).
It would be desirable to have an antithrombotic compound that can be activated to have an antithrombotic activity in the presence of a pathological blood clot which reduces or eliminates blood flow through a blood vessel, and which otherwise would be inactive, thereby reducing the risk of hemorrhagic complications.